Vietnam J. Agri. Sci. 2016, Vol. 14, No. 10: 1564 -1572<br />
<br />
Tạp chí KH Nông nghiệp Việt Nam 2016, tập 14, số 10: 1564 - 1572<br />
www.vnua.edu.vn<br />
<br />
ISOLATION AND IDENTIFICATION OF AN ACTINOMYCETE STRAIN<br />
WITH BIOCONTROL EFFECT AGAINST Xanthomonas oryzae pv. oryzae<br />
CAUSING BACTERIAL BLIGHT DISEASE IN RICE<br />
Nguyen Xuan Canh*, Phan Thi Trang, Tran Thi Thu Hien<br />
Faculty of Biotechnology, Vietnam National University of Agriculture<br />
Email*: nxcanh@vnua.edu.vn<br />
Received date: 05.08.2016<br />
<br />
Accepted date: 20.11.2016<br />
ABSTRACT<br />
<br />
In this study, we performed experiments to screen and identify actinomycete strains that are antagonistic to<br />
Xanthomonas oryzae pv. oryzae causing rice bacterial blight disease. Among of 98 strains isolated, we obtained two<br />
strains capable of antagonizing X. oryzae pv. oryzae using agar diffusion plate method. The strain numbered 43 had<br />
a strong activity with a diameter of 22 mm clear zone of bacteria. The strain 43 showed white colonies after three<br />
days of incubation. Seven days of incubation the white colonies had grey borders, produced soluble pigments on the<br />
medium, grew well at 30°C and neutral pH, and adapted well to high salt concentration medium. The strain 43 was<br />
able toutilize different sources of carbon and nitrogen. Sequence analysis of 16S rRNA showed that strain 43 had a<br />
similarity of 100% compared to Streptomyces diastaticus subsp. ardesiacus. Based on morphology, culture,<br />
physiological and biochemical characteristics and molecular biological analyses the strain 43 was identified as S.<br />
diastaticus subsp. ardesiacus.<br />
Keywords: 16S rRNA, Streptomyces sp., Xanthomonas oryzae pv. oryzae<br />
<br />
Phân lập và định danh chủng xạ khuẩn có khả năng<br />
đối kháng với vi khuẩn Xanthomonas oryzae pv. oryzae gây bệnh bạc lá lúa<br />
TÓM TẮT<br />
Trong nghiên cứu này chúng tôi đã tiến hành tuyển chọn, nghiên cứu đặc điểm sinh học của chủng xạ khuẩn có<br />
khả năng đối kháng với vi khuẩn Xanthomonas oryzae pv. oryzae gây bệnh bạc lá lúa. Từ 98 chủng xạ khuẩn có<br />
nguồn gốc khác nhau, bằng phương pháp khuếch tán thỏi thạch chúng tôi đã thu được 2 chủng có khả năng đối<br />
kháng với vi khuẩn X. oryzae pv. oryzae. Trong hai chủng thu nhận được thì chủng số 43 thể hiện hoạt tính mạnh<br />
hơn với đường kính vòng kháng khuẩn là 22 mm. Chủng 43 có khuẩn lạc màu trắng, nuôi từ 7 ngày trở đi thì có màu<br />
trắng viền xám, sinh sắc tố tan trên môi trường, sinh trưởng tốt ở nhiệt độ 30°C, pH trung tính và chịu được nồng độ<br />
muối tương đối cao tới 7%. Chủng 43 có khả năng sử dụng nhiều nguồn đường và nitrogen khác nhau. Phân tích<br />
trình tự 16S rRNA cho thấy chủng 43 và chủng Streptomyces aureofaciens có độ tương đồng là 100%. Kết hợp các<br />
đặc điểm hình thái, nuôi cấy, sinh lý, sinh hóa và phân tích sinh học phân tử đã xác định chủng xạ khuẩn 43 thuộc<br />
vào loài S. diastaticus subsp. ardesiacus.<br />
Từ khóa: 16S rRNA, Streptomyces sp., Xanthomonas oryzae pv. oryzae.<br />
<br />
1. INTRODUCTION<br />
Vietnam is one of the largest rice exporters<br />
in the world, however, the annual rice production<br />
is affected seriously by various diseases. The<br />
most serious disease is bacterial blight (BB)<br />
<br />
1564<br />
<br />
caused by Xanthomonas oryzae pv. oryzae (Xoo)<br />
(Gnanamanickam et al., 1999). The bacterial<br />
blight is distributed worldwide in most of rice<br />
producing countries and yield loss can be as<br />
much as 70% when susceptible varieties are<br />
grown in environments favorable to the disease.<br />
<br />
Nguyen Xuan Canh, Phan Thi Trang, Tran Thi Thu Hien<br />
<br />
This disease may attack the rice plants at the<br />
young<br />
stage but devastated mainly in the<br />
flowering period. The bacterium can penetrate<br />
through roots and clog vascular system but often<br />
resides and attacks on the leaves. Under high<br />
humidity or in<br />
rainy season, the disease<br />
becomes very severe. The disease causes dried<br />
leaves and decreases photosynthesis and<br />
therefore plants die prematurely or reduce yield.<br />
However, Xoo survives in vascular system,<br />
making it difficult to radically destroy without<br />
affecting the crop. Therefore research and<br />
application of methods to prevent as well as<br />
elimilate pathogens prior to each rice cropping<br />
season are neccessary so that farmers can reduce<br />
the outbreak or spread of BB.<br />
Integrated cultivation methods include field<br />
sanitation, eradication of weeds, adhering to the<br />
principles of intensive farming, proper<br />
fertilization, reasonable water level adjustment,<br />
and use of<br />
resistant varieties have been<br />
employed. In addition, seed and field chemical<br />
treatment before planting to eliminate the<br />
pathogen was highly effective but this may lead<br />
to the abuse of chemicals and environmental<br />
pollution and formation of chemical resistance<br />
of the pathogen. Therefore, it is essential to find<br />
out new compounds which are effectively in<br />
managing the disease but less harmful to<br />
environment and ecosystem as well.<br />
The Actinomycetes are known as a special<br />
group of bacteria which have the potential to<br />
generate a great deal of compounds that are<br />
able to kill bacteria and fungi. Scientists<br />
estimated that around 23,000<br />
compounds,<br />
which have biological activity, were produced<br />
from microorganisms, of which more than<br />
10,000<br />
compounds<br />
were<br />
isolated<br />
from<br />
actinomycetes (Watve et al., 2001). Therefore,<br />
actinomycetes are promising sources of<br />
bioactive substance production (Mitra et al.,<br />
2008). This study was carried out to screen and<br />
identify<br />
actinomycetes<br />
resistant<br />
to<br />
Xanthomonas oryzae pv. oryzae, since the<br />
search for new alternatives which are safe,<br />
efficient and environmental friendly in plant<br />
protection is of crucial significance.<br />
<br />
2. MATERIALS AND METHODS<br />
2.1. Materials<br />
The Xanthomonas oryzae pv. oryzae strain<br />
was isolated and stored at the Department of<br />
Molecular Biology and Applied Biotechnology,<br />
Faculty of Biotechnology, Vietnam National<br />
University of Agriculture.<br />
Around one hundred of actinomycete<br />
strains were isolated from various soil samples<br />
in Vietnam and stored at the Department of<br />
Microbial<br />
Biotechonology,<br />
Faculty<br />
of<br />
Biotechnology, Vietnam National University<br />
of Agriculture.<br />
2.2. Selection of actinomycete strains<br />
resistant to Xanthomonas oryzae pv. oryzae<br />
The actinomycete strains were plated on<br />
the Gause-1 medium (soluble starch 20 g/l;<br />
K2HPO4 0.5 g/l; MgSO4.7H2O 0.5 g/l, NaCl 0.5<br />
g/l; KNO3 0.5 g/l; FeSO4 0:01 g/l; Agar 20 g/l;<br />
pH = 7 to 7.4) at 30°C for five days. Cultured<br />
lumps of agar containing actinomycetes with 7<br />
mm in diameter were placed onto Wakimoto<br />
medium (300g potatoes, Ca(NO3)2.4H2O 0.5 g,<br />
Na2HPO4.12H2O 2 g, sucrose 15 g, Peptone 5 g,<br />
agar 20 g, water 1l, pH 7.0) containing<br />
Xanthomonas oryzae pv. oryzae, then incubated<br />
at 4°C for one hour to diffuse the active<br />
ingredients to the medium. The sample was<br />
transferred to 30°C incubator and observed<br />
after 12 hours of incubation, then the diameter<br />
of clear zone (if any) was measured.<br />
2.3.<br />
Identification<br />
the<br />
characteristics<br />
of<br />
the<br />
actinomycete (strain 43)<br />
<br />
biological<br />
selected<br />
<br />
The actinomycete strain numbered 43 was<br />
cultured on the Gause-1 medium at 30°C for<br />
five days and the morphology, color and size of<br />
the colonies were recorded.<br />
To identify spore chain morphology and<br />
spore surface the strain 43 was grown on the<br />
Gause-1 medium which was pinned by lamella<br />
with an angle of 45ºC. After 3 days of incubation<br />
at 30ºC, we drew out the lamella with aerial<br />
mycellium and observed spore chain morphology<br />
<br />
1565<br />
<br />
Isolation and identification of an actinomycete strain with biocontrol effect against Xanthomonas oryzae pv. oryzae<br />
causing bacterial blight disease in rice<br />
<br />
under an optical microscope. The morphology<br />
surface of actinomycete spores were observed<br />
under a scanning electron microscope (SEM).<br />
To check melanin pigmentation the strain<br />
43 was cultured on ISP-6 medium (Peptone 10<br />
g/l; yeast extract 1 g/l; iron citrate 0.5 g/l; Agar<br />
20 g/l; pH = 7.0 to 7.2) at 300C. The color of<br />
medium was observed for 21 days based onthe<br />
color change from yellow to brown or black.<br />
Checking the ability to assimilate carbon<br />
sources: the strain 43 was cultured on ISP-9<br />
medium ((NH4)2SO4 2, 64 g/l; KH2PO4 2.38 g/l;<br />
K2HPO4.3H2O 5.65 g/l ; MgSO4.7H2O 1 g/l; 1.0<br />
ml of solution B; Agar 20 g/l; pH = 6.8 to 7.0)<br />
supplemented with 1% by weight of the<br />
different sugar sources include D- glucose, Dfructose,<br />
D-manotol,<br />
sucrose,<br />
rhamnose,<br />
Inositol, L-arabinose, cellulose, D-xylose,<br />
raffinose. The ability to assimilate carbon<br />
sources was assessed by the viability and<br />
growth of actinomycetes on the medium.<br />
Checking the possibility of using nitrogen<br />
sources: the strain 43 was cultured on nitrate<br />
starch medium (Starch 20 g/l; NaNO3 2 g/l;<br />
K2HPO4 1 g/l; MgSO4.7H2O 0.5 g/l; KCl 0.5 g/l;<br />
FeSO4.5H2O 0:01 g/l; pH 6.8 - 7) as a control.<br />
The nitrogen sources include<br />
beef extract,<br />
KNO3, NH4Cl, peptone, (NH4)2SO4, NH4NO3 can<br />
be replaced with NaNO3.<br />
The effects of temperature, pH, and NaCl<br />
concentration on the growth and development of<br />
the strain 43 were determined by was culturing<br />
the strain on Gause-1 medium with the different<br />
culture temperatures (4, 20, 30, 40, 45, 50°C),<br />
pH (4 - 12) and NACl concentration (0 - 9%).<br />
2.4. Identification of the actinomycete<br />
strain 43<br />
Based on morphological characteristics and<br />
culture: the strain 43 was cultured on the<br />
medium and the<br />
morphology of colonies,<br />
substrate<br />
mycelium,<br />
aerial<br />
mycelium,<br />
conidiophore and surface of spore. The features<br />
identified<br />
were<br />
compared<br />
with<br />
known<br />
actinomycete strains<br />
in the international<br />
classification system (ISP) (Shirling and<br />
Gottlieb, 1966).<br />
<br />
1566<br />
<br />
Based on sequence analysis of 16S rRNA:<br />
DNA from the strain 43 was extracted by method<br />
described by Marmur (1961). PCR reaction<br />
amplified conservative regions of 16S rRNA with<br />
primers: 5'-AGAGTTTGATCCTGGCTCAG-3' and<br />
5'ACGGCTACCTTGTTACGACTT-3'.<br />
PCR<br />
products were checked on agarose gel 1%, and<br />
sent to company 1tsBASE (Singapore) for<br />
sequencing. The degree of similarity in the<br />
sequence of 16S rRNA<br />
of the strain 43 was<br />
compared with strains published in GenBank<br />
using<br />
Blast<br />
search<br />
tool<br />
(http://blast.ncbi.nlm.nih.gov/Blast .cgi). MEGA6<br />
software was used to determine genetic<br />
relationships. Maximum Parsimony selection<br />
method with reliability was calculated by<br />
bootstrap algorithm with 1000 repetitions. Based<br />
on the phylogentic tree and bootstrap values<br />
genetic relationship of the strain was determined.<br />
<br />
3. RESULTS AND DISCUSSION<br />
3.1. Screening of actinomycete strains<br />
antagonistic against Xanthomonas oryzae<br />
pv. oryzae<br />
The actinomycetes is supposed to be<br />
antagonistic against other microorganisms<br />
because of compounds with biological activities,<br />
especially many types of antibiotics. These<br />
substances are secreted to medium by<br />
actinomycetes during cultivation, so we used<br />
diffusion method on agar plates for selection and<br />
evaluation<br />
for<br />
antibacterial<br />
activity<br />
of<br />
actinomycetes. Gause-1 medium was used to<br />
grow the actinomycetes, Wakimoto medium was<br />
used to grow X. oryzae pv. oryzae. Through the<br />
screening, two of the 98 studied actinomycete<br />
strains were identified to be resistent to X.<br />
oryzae pv. oryzae. Among them, the strain<br />
numbered 43 was able to antagonize strongly<br />
with 22 mm of inhibited zone in diameter (Figure<br />
1 A), the strain numbered 978 had smaller<br />
inhibited zone with 16 mm in diameter (Figure 1<br />
B). In recent years, there have been some<br />
publications in the world in search for the<br />
actinomycetes capable of being antagonistic to X.<br />
oryzae pv. oryzae. These studies focused on the<br />
<br />
Nguyen Xuan Canh, Phan Thi Trang, Tran Thi Thu Hien<br />
<br />
discovery and extraction of active ingredients<br />
against Xoo as well as some other plant<br />
pathogens (Jiang et al., 2013; Kim et al., 2015;<br />
Park et al., 2011). Compared to the previous<br />
results, the strain 43 had relatively high activity.<br />
This result showed that the actinomycete strain<br />
43 had potential for application.<br />
3.2. Biological characteristics<br />
actinomycete strain 43<br />
<br />
of<br />
<br />
the<br />
<br />
3.2.1. Morphological characteristics<br />
One of the first criteria to study biological<br />
characteristics<br />
and<br />
classification<br />
of<br />
actinomycetes is based on morphological<br />
characteristics (Miyadoh et al., 2016). The<br />
actinomycete strains were grown on Gause-1<br />
medium at 30°C for 7 days to observe the color,<br />
size, shape of the colony. After three days of<br />
culture, the colony of strain 43 showed round<br />
shape, 0.2 - 0.4 mm in size with off-white color.<br />
The color of colony changed after some days of<br />
incubation and colonies were almost dark brown<br />
at day 5.<br />
After determining the characteristics of<br />
colonies, we determined the formation of<br />
conidiophore, spore chain and surface of spore<br />
from the strain 43. The results observed under<br />
<br />
A<br />
<br />
an optical microscope at a magnification of 1000<br />
showed that after 48 h of incubation the<br />
actinomycetes strain 43 started sporulation.<br />
The spores arranged in long, branched and<br />
twisted chains. After 60 hrs of culture, the<br />
spores began to leave the series and released to<br />
the<br />
culture medium. To determine more<br />
accurately the morphology of the strain 43, we<br />
observed the morphology of the spore chain and<br />
the surface of spore under a scanning electron<br />
microscope<br />
(SEM).<br />
Specimen<br />
handling,<br />
observation and analysis of the image were<br />
carried out in the National Institute of Hygiene<br />
and Epidemiology. Using SEM with 5000 times<br />
magnification, it showed that the spore chain of<br />
the strain 43 was typical with a white spiral<br />
spring form, each chain bearing 10 - 20 spores<br />
(Figure 2 A). Spores of the strain 43 had short<br />
oval shape with size of 0.6 - 0.8 x 0.9 - 1.1 µm<br />
and smooth surface (Figure 2 B).<br />
3.2.2. Ability of melanin pigmentation<br />
According to the International Streptomyces<br />
Project (ISP), melanin formation was determined<br />
on the ISP6 medium at 30°C for at least 21 days.<br />
Melanin production changed<br />
the color of<br />
medium from pale yellow to brown and black<br />
(Shirling and Gottlieb, 1966).<br />
<br />
B<br />
<br />
Figure 1. Antagonistic activity of some actinomycetes against Xanthomonas oryzae pv.<br />
oryzae by diffusion method on agar plates. Two strains of actinomycete strains<br />
identified as active strains are strain 43 (A) and strain 978 (B)<br />
<br />
1567<br />
<br />
Isolation and identification of an actinomycete strain with biocontrol effect against Xanthomonas oryzae pv. oryzae<br />
causing bacterial blight disease in rice<br />
<br />
(A)<br />
<br />
(B)<br />
<br />
Figure 2. Mophology of spore chain and spore surface from the strain 43 under scanning<br />
electron microscope at magnification of 5,000 times (A) and 3,0000 times (B)<br />
<br />
(A)<br />
<br />
(B)<br />
<br />
Figure 3. Melanin formation of the strain 43 when cultured on the ISP-6 medium<br />
after 21 days. Photographs of the up-side (A) and bottom-side (B) of cultured dish<br />
Table 1. Ability of using various carbon and nitrogen sources of strain 43<br />
<br />
Fructose<br />
<br />
Development of the strain 43<br />
after 5 days of culture<br />
+<br />
<br />
NaNO3<br />
<br />
Mantose<br />
<br />
+<br />
<br />
KNO3<br />
<br />
+<br />
<br />
Xylose<br />
<br />
+<br />
<br />
Beef extract<br />
<br />
++<br />
<br />
Arabinose<br />
<br />
+<br />
<br />
NH4Cl<br />
<br />
-<br />
<br />
Rhanbinose<br />
<br />
++<br />
<br />
Pepton<br />
<br />
+<br />
<br />
Carbon source<br />
<br />
Nitrogen source<br />
<br />
Development of the strain 43<br />
after 5 days of culture<br />
+<br />
<br />
Sucrose<br />
<br />
-<br />
<br />
(NH4)2SO4<br />
<br />
-<br />
<br />
Lactose<br />
<br />
+<br />
<br />
NH4NO3<br />
<br />
-<br />
<br />
Manitol<br />
<br />
++<br />
<br />
Yeast extract<br />
<br />
Sobitol<br />
<br />
-<br />
<br />
Dextrose<br />
Ribose<br />
Galactose<br />
<br />
++<br />
++<br />
<br />
Note: (+) the strain 43 could grow; (++) the strain 43 grow develop well; (-) the strain 43 cannot grow<br />
<br />
1568<br />
<br />
++<br />
<br />